Inhibitory effects of calcitriol on the growth of MCF-7 breast cancer xenografts in nude mice: selective modulation of aromatase expression in vivo

Abstract

Calcitriol (1,25-dihydroxyvitamin D(3)), the hormonally active metabolite of vitamin D, exerts many anticancer effects in breast cancer (BCa) cells. We have previously shown using cell culture models that calcitriol acts as a selective aromatase modulator (SAM) and inhibits estrogen synthesis and signaling in BCa cells. We have now examined calcitriol effects in vivo on aromatase expression, estrogen signaling, and tumor growth when used alone and in combination with aromatase inhibitors (AIs). In immunocompromised mice bearing MCF-7 xenografts, increasing doses of calcitriol exhibited significant tumor inhibitory effects (~50% to 70% decrease in tumor volume). At the suboptimal doses tested, anastrozole and letrozole also caused significant tumor shrinkage when used individually. Although the combinations of calcitriol and the AIs caused a statistically significant increase in tumor inhibition in comparison to the single agents, the cooperative interaction between these agents appeared to be minimal at the doses tested. Calcitriol decreased aromatase expression in the xenograft tumors. Importantly, calcitriol also acted as a SAM in the mouse, decreasing aromatase expression in the mammary adipose tissue, while increasing it in bone marrow cells and not altering it in the ovaries and uteri. As a result, calcitriol significantly reduced estrogen levels in the xenograft tumors and surrounding breast adipose tissue. In addition, calcitriol inhibited estrogen signaling by decreasing tumor ERα levels. Changes in tumor gene expression revealed the suppressive effects of calcitriol on inflammatory and growth signaling pathways and demonstrated cooperative interactions between calcitriol and AIs to modulate gene expression. We hypothesize that cumulatively these calcitriol actions would contribute to a beneficial effect when calcitriol is combined with an AI in the treatment of BCa.

Keywords: Aromatase; Aromatase inhibitors; Breast cancer; Calcitriol; Estrogen synthesis; Selective aromatase modulator; Xenografts.

Fig. 1

Effect of calcitriol, AIs, and their combinations on the growth of MCF-7 tumors. MCF-7 xenografts were established in the flanks of intact nude mice (premenopausal model). After 6 weeks of tumor growth the mice were given i.p. injections of calcitriol at very low (VLC, 0.025 μg/mouse), low (LC, 0.05 μg/mouse), and high (HC, 0.1 μg/mouse) concentrations three times a week for the following 4 weeks. Tumor volumes were measured weekly and calculated as described in Materials and Methods (a). Some mice received anastrozole (Ana, 5 μg/mouse, s.c. or i.p. injections 6 days a week) as a single agent or in combination with LC (LC + Ana) and the tumor volumes were measured over the next 4 weeks (b). In separate experiments, MCF-7 xenografts were established in right and left fourth mammary fat pads of OVX nude mice (postmenopausal model) implanted with 60-day time-release androstenedione pellets and allowed to grow for 6 weeks. The mice received very low dose calcitriol (VLC, 0.025 μg/mouse, three times a week as i.p. injections), letrozole (Let, 2.5 μg/mouse, s.c. or i.p. injections 6 days a week) or a combination of both (VLC + Let) and the tumor volumes were measured over the next 4 weeks (c). Values represent mean ± SE. Number of mice in the various experimental groups were as follows: In a and b, control = 17, VLC = 6, LC = 10, HC = 8, Ana = 17, and LC + Ana =18. In c, control = 13, VLC = 6, Let = 13, and VLC + Let = 12. a = p < 0.001 as compared to control, b = p < 0.05 as compared to VLC, c = p < 0.05 as compared to LC, and d = p < 0.05 as compared to Ana

Fig. 2

Tissue-specific regulation of aromatase expression by calcitriol. (a) Total aromatase mRNA levels were measured by qRT-PCR as described in Materials and Methods in tumors, ovaries, uteri, mammary fat, and bone marrow cells from intact mice receiving low dose calcitriol (LC) treatment as described in Fig. 1. Relative aromatase mRNA levels in control mice was set at 1 for each tissue. Values represent mean ± SE from six to 11 determinations. *p < 0.05 and **p < 0.01 as compared to control. (b) Total aromatase mRNA levels were measured in adipose tissue from non- tumor-bearing and tumor-bearing mammary fat pads from OVX mice receiving very low dose calcitriol (VLC) treatment as described in Fig. 1. Values represent mean ± SE from 6 to 11 determinations. ⁺ p < 0.05 when the levels in tumor bearing adipose tissue in control mice were compared to the levels in non-tumor-bearing adipose tissue in control mice. **p < 0.01 when the levels in the non-tumor-bearing and tumor bearing mammary fat in mice receiving VLC were compared to the corresponding control mice receiving vehicle

Fig. 3

Changes in tumor gene expression due to calcitriol, AI, and their combinations. Vehicle (control, Con), calcitriol, AIs, and their combinations were administered to tumor bearing intact (a and b) or OVX (c and d) mice as described in Fig. 1. Total RNA was isolated from harvested tumors and the mRNA levels of ERα, aromatase COX-2, p21, and IGFBP-3 were determined by qRT-PCR as described in Materials and Methods. Relative mRNA expression for each gene in tumors from control mice was set at 1. Values represent mean ± SE from six to 11 determinations. ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001 as compared to control. ⁺ p < 0.05 and ⁺⁺ p < 0.01 as compared to LC or VLC. ^ p < 0.05 and ^^ p < 0.01 as compared to Ana or Let

Fig. 4

Effect of calcitriol, anastrozole, and their combinations on ERα and aromatase protein expression in tumors. Vehicle (control, Con), low dose calcitriol (LC), anastrozole (Ana), and their combination (LC + Ana) were administered to intact mice as described in Fig. 1. Tumors were harvested and fixed for IHC analysis of aromatase and ERα expression as described in Materials and Methods. All tumors were immunostained with either anti-ERα antibody (a–d) or an anti-aromatase antibody (e–h) and are shown at 400× magnification. i Immunostaining with IgG (negative control). Numbers 0 to 3+ indicate the intensity of staining and numbers followed by the % sign indicate percentage of cells showing positive staining

Fig. 5

Effects of calcitriol, letrozole, and their combination on E₂ and E₁ levels in tumors and surrounding mammary adipose tissue. Vehicle (control, Con), very low dose calcitriol (VLC), letrozole (Let), and their combination (VLC + Let) were administered to OVX mice as described in Fig. 1. Tumors and surrounding mammary adipose tissue were harvested from the mice and E₂ (a) and E₁ (b) levels in the tissue extracts were determined as described in Materials and Methods. Values represent mean ± SE from five to 10 determinations. ^** p < 0.01 and ^*** p < 0.001 as compared to control. ⁺ p < 0.05 as compared to VLC. ^^ p < 0.01 as compared to Let